Calculations of the metabolic flux through enzymes in vivo produce results which are sometimes incompatible with observed rates. One possible basis for these discrepancies is that the values of the kinetic constants used are different from those of the enzymes in situ. This has been shown experimentally; the Km of ATPMg for mitochondrial carbamyl phosphate synthetase in situ is much lower than that for the enzyme in dilute solution. Another difference, suggested by indirect evidence, is that ornithine greatly stimulates carbamyl phosphate synthetase in situ, while it has little effect on this enzyme in dilute solution. If, as appears to be the case, such discrepancies exist in the behavior of other enzymes, the kinetic characterization of enzymes in situ would be essential for understanding some aspects of metabolic regulation in vivo. The ultimate goal of this research is to obtain physiologically valid enzymological information pertaining to the regulation of metabolism in vivo in both, normal and pathological states. Procedures have been developed to study the kinetic properties of carbamyl phosphate synthetase (ammonia) and ornithine transcarbamylase in situ, using isolated liver mitochondria and mitoplasts incubated under conditions in which the concentration of a single reactant can be varied and known. This will be achieved by the use of uncouplers, inhibitors, and ionophores, as required. Experiments will be designed to determine the apparent Km values of carbamyl phosphate synthetase for ammonia and free Mg2+, and those of ornithine transcarbamylase for carbamyl phosphate and ornithine. Studies on the stimulation of carbamyl phosphate synthesis by ornithine will be continued in order to establish the mechanism of this effect. This work will provide basic data on enzyme behavior in a physiological environment; the information derived should be relevant to disciplines dealing with the biophysical chemistry of proteins, enzyme kinetics, metabolic regulation, and the in vivo organization of macromolecules.